Fibrinogen is an essential protein for the blood coagulation because its polymerisation to insoluble fibrin, which is formed at the end of the reaction cascade governing the coagulation, leads to the formation of a clot, blocking the vascular gap, responsible for the bleeding. The formation of the clot is essential to stop the bleeding. Further, the fibrin formed on the wound level forms a fibrillary network ensuring the tissue repair (wound healing).
Congenital fibrinogen deficiencies can lead to serious diseases. In order to treat these deficiencies, it is necessary that fibrinogen concentrates, which can be administered to patients under treatment, are available. Other pathologies can also be treated by administering of fibrinogen, especially in cases of massive blood losses (surgery, traumas, etc.), or following to a disseminated intravascular coagulation syndrome (CIVD).
Moreover, biological glues, activated by thrombin, containing fibrinogen as the major component, and Factor XIII (FXIII), are efficiently used in tissue repair in clinics, such as skin transplantation, nervous and arterial sutures, as described, for example, in patents EP 0 305 243, FR 2 448 900 and FR 2 448 901. The presence of Factor XIII or transglutaminase in these products contributes to the stabilization of fibrin by formation of intercatenary covalent bindings which make it insoluble. In some cases, these products are obtained by means of rather complex fibrinogen production processes, which require an external supply of purified Factor XIII, in order to be able to perform their therapeutic function.
Therefore, production of fibrinogen, biological glues and Factor XIII concentrates, especially for therapeutic uses, requires purification techniques leading to these products, which are not only sufficiently free of various contaminants, such as the accompanying or co-precipitated proteins, antibodies or proteases but, in addition, their viral safety is increased.
The isolation of fractions enriched in fibrinogen, possibly containing FXIII, from plasma, is well known and first described by Cohn and Nitschmann (Cohn et al, J. Am. Chem. Soc., 68, 459, 1946 and Kistler et al, Vox Sang., 7, 1962, 414-424). More recent methods combine precipitating techniques of different plasma sources with filtration, chromatography, viral inactivation techniques, etc. The following patents and patent applications can be cited as examples: EP 0 359 593, U.S. Pat. No. 5,099,003, EP 0 305 243, FR 2 448 900 and FR 2 448 901.
Nevertheless, different processes yielding concentrates or compositions either of fibrinogen, as described in the patent application EP 1 457 497, or of biological glue, for example according to the patent EP 0 771 324, or enriched in fibrinogen containing further associated proteins such as FXIII, Factor VIII, fibronectin, Factor von Willebrand etc. (especially U.S. Pat. No. 6,121,232) are carried out.
These processes, however, involve the use of separate production lines consequently using different methods employing several sources of raw materials for obtaining these considered proteins. Furthermore, depending upon the case, these methods can involve expensive chromatographic substrates, such as affinity gels based on chelated metals (WO 2004/007533) liable to release residual metals into the eluate, which can lead to unwanted reactions with the proteins (for example oxidation). This creates problems of clumsiness of the carrying out on industrial scale, when these three purified active principles are needed together. These problems are even more obvious when the different thus obtained proteins are to be subjected to a viral inactivation and/or viral and other unwanted contaminants, such as prions, removal treatment.
To this end, some classical viral inactivation treatments implementing a heat treatment, such as pasteurisation at 60° C., for 20 hours, in the presence of protecting stabilizers, and a chemical treatment, such as solvent-detergent, intended to make the above concentrates compatible with therapeutic use, do not allow to inactivate completely the viruses, especially non-enveloped viruses (parvovirus B19, hepatitis A and B, etc.).
In order to find a solution to this drawback, use is currently made of more efficient viral inactivation processes, such as dry heat treatment under harsh conditions (80° C., 72 h). This step requires the incorporation of a suitable stabilizing formulation offering conditions such as, for example, the fibrinogen stabilization in this step, while the viruses are being destroyed. Such a formulation is the subject-matter of a patent application FR 04 02001 filed by the Applicant. However, this formulation can be applied to the stabilization of a defined protein and not of the accompanying proteins, characteristics of which are different of those of fibrinogen.
The filtration techniques, especially the nanofiltration using filters with a porosity of 35 nm, and even less, have also been carried out in order to remove viruses. However, this technique cannot be efficiently used without controlling the physical and chemical parameters influencing the recovery output of compounds to be filtered, and this by avoiding the clogging of the filter and the passage of various viruses and contaminants. These parameters, such as ionic strength, pH of the solution, and filtration process conditions, as well, lay down the specific process conditions which depend also on the nature of the compound(s) contained in the solution to be filtered. Although the patent applications EP 1 348 445 A1, EP 1 161 958 A1 and WO 99/23111 disclose the nearly total removal of very small sized non-enveloped viruses present in the protein solutions, such as hepatitis A, by nanofiltration, making use of filters of 15 nm, however, the risk of transmission of unwanted viruses or prions is always present.
In order to avoid this risk, a double or even a triple viral inactivation and/or removal combining at least two of the above mentioned techniques can be performed, as described for example in the patent application WO 2004/007533. When such treatments are combined, then it is essential to choose, depending upon the viral inactivation method, the virucidal excipients and/or protecting stabilizers which are not exerting a concomitant deleterious effect, as for example on the above mentioned physical and chemical parameters governing the nanofiltration.